A Direct Immersion System for Peptide Enrichment

Abstract

A complementary peptide separation and purification system, using a novel, artificial, affinity-type extraction material, is described. Cellulose paper was first coated with a 3-methacryloxypropyltrimethoxysilane (MPS) layer, followed by imprinting with peptides, to form an approximately 2-µm layer of film (molecularly imprinted polymer film; MIPF) on both sides and resulted in an increased affinity toward the corresponding template as well as to matched polypeptides. MIPF-cellulose paper (MIFC) was immersed in 2% peptide solution (acetonitrile/water) for the sorption of targeted-peptides at room temperature for an hour in a 96-well microtiter plate. Desorption of targeted-peptides from the MIPF-and NIPF (non-imprinted polymeric film)-coated filter papers was nearly complete (~99%) in two desorptions of 10 min using 5% acetic acid in water. Clean extracts of targeted-peptides were obtained demonstrating the suitability of MIPF-coated filter papers for the extraction or analysis of biological samples. To compare their extraction characteristics under various conditions, analyses were carried out by an enzyme-linked immunosorbent assay (ELISA) microplate reader as peptide-detecting/peptide-capturing microarrays. In load ability studies using acetonitrile/water, peptide masses up to 100 µg could be applied onto a piece of MIPF coated filter paper (0.7 cm in diameter). ArticleTai et al. Scheme II Operation of MIFC for the extraction of peptide Fig. 2. Scatchard plot for the binding of the Phe-Phe-Phe-Phe to MIFC. Data points are the average of triplicates.

Exploring the peptide retention mechanism in molecularly imprinted polymers

“…This has to take in account many different interactions, simultaneously occurring around the template molecule, and the template insolubility into organic solvents generally used for the polymeric synthesis [26][27] . However, several MIPs have been developed for peptide enrichment and employed in the sample preparation of proteomic workflows [28][29][30][31][32][33][34][35] .…”

Peptide conformational imprints enhanced the catalytic activity of papain for esterification

“…Then, 200 µl of the supernatant was taken out and measured using a Fluorescence Microplate Reader at E ex / E em = 290 nm/350 nm. The binding affinities of PCIMPs were evaluated using the Scatchard analysis equation ( 1 ) ( Gerdon et al, 2005 ; Diltemiz et al, 2009 ; Tai et al, 2012 ; Kanubaddi et al, 2021 ), …”

“…The PCIMPs-grafted 15-and 14-mer peptides were then tested for their ability to bind their mother protein, as described previously (Tai et al, 2012). As shown in Table 2, it was found that PCIMPs 65-79 had more significant binding affinities, while the other two, PCIMPs [65][66][67][68][69][70][71][72][73][74][75][76][77][78] and PCIMPs 66-79 , exhibited lower affinity.…”

Deviation of Trypsin Activity Using Peptide Conformational Imprints

“…Then, 200 µL of supernatant was collected and measured by Fluorescence Microplate Reader at λex/λem = 290 nm/350 nm. Each experiment was repeated three times, and the results of the binding studies were evaluated using the Scatchard Equation (1) [ 27 , 28 , 29 ]. [RL]/[L] = (B max − [RL])/K d where [L] is the concentration of PPT in the solution, [RL] is the concentration of bound PPT, B max denotes the maximum number of binding sites, and K d is the dissociation constant of the ligand.…”

“…Then, 200 µL of supernatant was collected and measured by Fluorescence Microplate Reader at λex/λem = 290 nm/350 nm. Each experiment was repeated three times, and the results of the binding studies were evaluated using the Scatchard Equation (1) [27][28][29].…”